The pyruvate dehydrogenase complex regulates mitophagic trafficking and protein phosphorylation

Mutations in the PDC affect the phosphorylation and mitophagic trafficking of matrix proteins, through the novel regulation of associated kinases and a phosphatase. We suggest that this occurs by the direct allosteric regulation of the phosphatase and kinases by the PDC.


Introduction
Mitochondrial autophagy, or mitophagy, is a quality control mechanism that culls defective mitochondria in an effort to prevent cellular damage and dysfunction. Indeed, defects in mitophagic mechanisms have been shown to underlie human pathologies that can be traced to mitochondrial dysfunction. These include Parkinson's disease, Alzheimer's disease, and type II diabetes (Vafai & Mootha, 2012;Ploumi et al, 2017;Metaxakis et al, 2018). Mitophagy has also been shown to be important for developmental transitions in which the mitochondrial proteome needs to be drastically remodeled (Schweers et al, 2007;Sin et al, 2016;Esteban-Martínez et al, 2017). Known mitophagic mechanisms all involve a receptor protein, which interacts with mitochondria on the one hand, and engages the autophagic machinery via LC3/ Atg8 proteins and ancillary factors in response to specific cues on the other hand (Abeliovich & Dengjel, 2016). In mammalian cells, known mitophagy receptors include outer mitochondrial membrane cardiolipin, NIX, BNIP3, NDP52, optineurin, prohibitin 2, TAX1BP1, FUNDC1, and Bcl2-L-13 (Schweers et al, 2007;Bellot et al, 2009;Novak et al, 2010;Liu et al, 2012;Lazarou et al, 2015;Murakawa et al, 2015;Wei et al, 2017), all of which function under different induction conditions and in different cell types. In budding yeast cells, a single outer mitochondrial membrane protein, Atg32, functions as the only known mitophagy receptor (Kanki et al, 2009;Okamoto et al, 2009). Current thinking envisions the selective activation of a mitophagy receptor on specific mitochondria, which leads to specific engulfment and degradation of those mitochondria. Thus, it is surprising that mitophagic processes, from yeast to humans, exhibit protein-level selectivity. We previously demonstrated, using SILAC-based proteomics and additional analyses, that different mitochondrial matrix proteins exhibit different mitophagic trafficking rates in yeast and that this phenomenon correlates with physical segregation of the different proteins within the mitochondrial network (Abeliovich et al, 2013). Similar observations were reported in human cells (Hämäläinen et al, 2013;Burman et al, 2017) and in Drosophila (Vincow et al, 2013(Vincow et al, , 2019. More recently, we found that protein phosphorylation in the mitochondrial matrix plays a role in determining intra-mitochondrial mitophagic selectivity. Briefly, we discovered that mitophagic trafficking of the reporter protein Mdh1-GFP depends on its phosphorylation status and that phosphorylation of the relevant residues requires the function of the mitochondrial matrix kinases Pkp1 and Pkp2, acting under the regulation of the Aup1/Ptc6 phosphatase, which had previously been implicated in mitophagy (Tal et al, 2007;. Pkp1, Pkp2, and Aup1 have also been shown to regulate the phosphorylation of Pda1, the yeast E1α subunit of the mitochondrial pyruvate dehydrogenase complex (PDC) (Krause-Buchholz et al, 2006;Gey et al, 2008;Guo et al, 2017a). One simple explanation for the data would be that the pyruvate dehydrogenase reaction, which generates acetyl-CoA from pyruvate in the mitochondrial matrix, is required for mitophagy or for mitophagic selectivity. However, several problems arise with this hypothesis. For example, in the regulation of pyruvate dehydrogenase activity, Pkp1 and Pkp2 function antagonistically relative to Aup1: they are required for phosphorylation of S313 on Pda1, which inactivates enzymatic activity, whereas Aup1 mediates dephosphorylation of this residue and activation of the enzyme (Guo et al, 2017a). In contrast, in the regulation of Mdh1-GFP mitophagic trafficking, Pkp2 and Pkp1 function together with Aup1: both the kinases and the phosphatase are required for efficient mitophagy of several reporters, with the kinases functioning downstream of Aup1, as Pkp2 overexpression suppresses the aup1Δ phenotype .
To clarify and reconcile the relationship between the functions of Pkp1, Pkp2, and Aup1 in mitophagy versus their function in Pda1 regulation, we have now examined the potential role of the PDC in generating mitophagic selectivity. Surprisingly, the results of this investigation define a novel signaling cascade that is regulated by the PDC, but is uncoupled from its known enzymatic activity.

Results
Deletion of PDA1 has a marked effect on mitophagic selectivity, which is uncoupled from pyruvate dehydrogenase activity Because Aup1, Pkp1, and Pkp2 have been identified as pyruvate dehydrogenase kinase phosphatase and kinases (Krause-Buchholz et al, 2006;Gey et al, 2008;González et al, 2013;Guo et al, 2017a), we wanted to understand the relationship between their role in regulating mitophagic selectivity, and their role in regulating pyruvate dehydrogenase activity. Surprisingly, we found that deletion of the PDA1 gene, which encodes the E1α subunit of the PDC, has a major negative impact on the mitophagic trafficking of five different mitochondrial matrix proteins (Fig 1) when measured using the free GFP release assay. In contrast, the pda1Δ mutation had no effect on the mitophagic trafficking of an artificial mitophagy reporter, mtDHFR-GFP, as we previously reported for the pkp1Δ pkp2Δ double mutant . This result implies that the pda1Δ mutation does not block mitophagy per se, but rather affects the sorting of individual proteins to a mitophagy-targeted mitochondrial subpopulation, based on native protein-protein interactions that are not accessible to the artificial DHFR-based reporter. In addition, deletion of PDA1 had no effect on the levels of Pkp1, Pkp2, or Aup1 ( Fig S1).
The most straightforward explanation for this result would be that the pyruvate dehydrogenase reaction itself, i.e. the formation of acetyl-CoA from pyruvate with concomitant reduction of NAD + , is required for efficient mitophagy. However, deletion of the mitochondrial pyruvate carrier subunit MPC1 had no effect on mitophagic trafficking (Fig 2A and B), even though this mutation prevents entry of pyruvate into mitochondria (Herzig et al, 2012). We also tested whether the role of Aup1 in mitophagy is limited to the dephosphorylation of Pda1 at position Ser313 (the residue regulated by Pkp1/2 and Aup1 [Gey et al, 2008;Guo et al, 2017a]). To this end, we determined whether deletion of AUP1 can affect mitophagic trafficking in cells where the sole copy of PDA1 encodes a serine to alanine mutation at position 313. As shown in Fig 2C and D, the Pda1 S313A mutant can weakly complement deletion of the WT protein for mitophagic trafficking of the Mdh1-GFP reporter. However, this residual mitophagic trafficking is completely lost in cells which also lack Aup1, indicating that Aup1 regulates mitophagic trafficking of Mdh1 independently of the phosphorylation state of serine 313 on Pda1. Finally, we tested whether catalytically inactive point mutations in the E1 and E2 subunits affect the mitophagic trafficking of the Mdh1-GFP reporter. For the E1α subunit Pda1, we chose the R322C mutation, which was previously shown to completely abrogate catalytic activity (Drakulic et al, 2018). As shown in Fig 2E and F, although this point mutation caused a significant decrease in mitophagic trafficking of Mdh1-GFP, it still allowed a reproducible signal of free GFP under mitophagy-inducing conditions, which was significantly higher than that observed in the pda1Δ deletion, and does not phenocopy the deletion. More importantly, when we introduced the unlipoylatable K75R mutation in the E2 subunit Lat1 (Lawson et al, 1991;Gey et al, 2014), the expression of this catalytically inactive mutant subunit led to a clear increase in mitophagic trafficking of Mdh1-GFP-completely opposite from the result expected if the lat1Δ phenotype was the result of a block in PDC catalytic activity (Fig 2G and H). Neither lat1Δ nor lat1 K75R had any effect on the mitophagic trafficking of mtDHFR-GFP ( Fig S2). Collectively, these results indicate that the effects of the pda1Δ and lat1Δ mutations on mitophagic trafficking cannot simply be explained by the loss of PDC enzymatic activity, and are uncoupled from the phosphorylation state of serine 313 on Pda1.
The PDC affects phosphorylation of Mdh1-GFP via regulation of Pkp1 and Pkp2 The extent of the decrease in mitophagic trafficking of mitochondrial matrix GFP chimeras, which we observe in pda1Δ cells ( Fig  1A), is very similar to the phenotype which we had previously reported for the pkp1Δ pkp2Δ double mutant . Those previous studies showed that Mdh1-GFP is strongly hypophosphorylated in pkp1Δ pkp2Δ double mutants ). An alternative hypothesis that we therefore wanted to explore was that the PDC reciprocally regulates its associated kinases and phosphatases, by virtue of their physical association (Krause-Buchholz et al, 2006;Breitkreutz et al, 2010;Guo et al, 2017a), as such interactions with the PDC could potentially regulate the enzymatic activity of Aup1, Pkp1, and Pkp2. If the effect of the pda1Δ mutation reflects the reciprocal regulation of Aup1, Pkp1, and Pkp2 by the PDC, we would expect that the loss of Pda1 would similarly affect the phosphorylation of Mdh1-GFP. Indeed, we find that Mdh1-GFP phosphorylation is strongly attenuated in pda1Δ cells and that this phenotype can be complemented by expressing Pda1 from a plasmid (Fig 3A and B). Similarly, knockout of PDA1 also led to hypophosphorylation of Qcr2-GFP and Aco1-GFP under these conditions (Figs S3 and S4). It was previously shown that Aup1 and Pkp1 stably interact with the PDC (Krause-Buchholz et al, 2006;Breitkreutz et al, 2010;Guo et al, 2017a). To determine whether the PDC might function upstream of the kinases in regulating mitophagic trafficking of Mdh1-GFP, we tested whether the overexpression of Pkp1, Pkp2, or both kinases together might rescue the mitophagy defect observed in pda1Δ cells. As shown in Fig 3C and D, the co-overexpression of Pkp1 together with Pkp2, but not each kinase separately, caused a near-complete rescue of the pda1Δ mitophagy phenotype. Importantly, deletion of PDA1 did not affect the levels of Pkp1, Pkp2, or Aup1 (Fig S1), indicating that the loss of Pda1 does not destabilize these proteins or otherwise affect their expression. In addition, we were able to show a similar suppression pattern for the overexpression of Pkp1 and Pkp2 in aup1Δ cells, supporting the hypothesis that the PDC regulates Pkp1 and Pkp2 via an effect on Aup1 (Fig 3E and F).
We previously showed that the mitophagy phenotype of the pkp2Δ mutation could be suppressed in cells expressing the Mdh1 T199D -GFP reporter, which mimics phosphorylation of Mdh1 at residue 199 . If the effects of the pda1Δ mutation are due to an effect of the PDC on Pkp1 and Pkp2, then we expect that, similarly, the Mdh1 T199D -GFP reporter would show at least partial suppression in the pda1Δ background. As shown in Fig  3G and H, the expression of the Mdh1 T199D -GFP variant, but not the Mdh1 T199A -GFP mutant, efficiently bypassed the Mdh1 mitophagic trafficking defect block in the pda1Δ mutant. Taken together, these results strongly support the argument that Pda1 affects mitophagy by regulating the phosphorylation state of the reporter.
Furthermore, the co-overexpression of Pkp1 and Pkp2 increases the phosphorylation of Mdh1-GFP in pda1Δ cells, as would be expected if the PDC was modulating Pkp1/2 activity toward downstream targets (Fig 4A and B), again supporting our finding that it is not PDC catalytic activity, which is important for the mitophagic trafficking of Mdh1-GFP.
We were also able to demonstrate a clear physiological function for Pkp1 and Pkp2, which is uncoupled from the phosphorylation of the E1α subunit: although pda1Δ cells show a marked reduction in the percentage of colony-forming cells after prolonged incubation in respiratory medium, the co-overexpression of Pkp1 and Pkp2 fully suppressed this phenotype (Figs 4C and S5). This result WT (HAY75) and pda1Δ (PKY832) cells expressing the indicated GFP-tagged mitochondrial matrix proteins were grown in SL medium as indicated in the "Materials and Methods section." Samples were taken on days 1 and 5, and protein extracts were analyzed by anti-GFP immunoblotting to determine the percent of signal converted to free GFP. (A) Loss of PDA1 strongly attenuates mitophagic trafficking of Mdh1-GFP, and this can be complemented by expressing Pda1 from a plasmid. (B) Loss of PDA1 strongly attenuates the mitophagic trafficking of endogenous GFP chimeras of five different mitochondrial matrix proteins, but not of the "artificial" mtDHFR-GFP reporter. (B, C) Representative immunoblots for the data summarized in (B). Data are shown from three independent biological replicates ± SD. ϕ denotes an empty vector. demonstrates that the two kinases have a physiological function other than the regulation of PDC catalytic activity, and at the same time, the epistasis relationships indicate-like our other resultsthat the PDC seems to act as an upstream regulatory element in the(se) novel signaling pathway(s), independent of its catalytic activity. If Pda1 regulates Mdh1-GFP phosphorylation via regulation of the activity of Pkp1 and Pkp2, then co-deletion of the opposing phosphatase should partially reverse this defect, as any residual phosphorylation activity would now be amplified by the loss of the opposing phosphatase activity. Our previous work demonstrated that Aup1/Ptc6 cannot be opposing Pkp1/Pkp2 in regulating mitophagic trafficking, because (1) deletion of AUP1 leads to a phenotype that is similar to the phenotypes of pkp1Δ and pkp2Δ on mitophagic trafficking, and (2) Aup1/Ptc6 functions upstream of Pkp1 and Pkp2 in regulating Mdh1-GFP phosphorylation and mitophagic trafficking . We therefore tested the effects of deleting the mitochondrial phosphatases PTC5 and PTC7 (Guo et al, 2017a; 2017b) on Mdh1-GFP mitophagic trafficking, in the absence of Pda1. As shown in Fig 4D and E, both deletion of PTC5 and, to a much lesser extent, deletion of PTC7 led to some generation of free GFP in pda1Δ cells, whereas the phenotype of the combined ptc5Δ pdc7Δ double deletion seems identical to the ptc5Δ phenotype. This result further supports the hypothesis that the PDC regulates the phosphorylation state of Mdh1-GFP, and suggests that Ptc5 is the main mitochondrial phosphatase, which antagonizes the function of Pkp1 and Pkp2 in regulating mitophagic trafficking of Mdh1-GFP (see the Discussion section).
To further test the possibility that conformational changes in the PDC may regulate Pkp1 and Pkp2 activity, we asked whether the catalytically inactive lat1 K75R mutation, which increases mitophagic efficiency of Mdh1-GFP ( Fig 2G and H), also increased phosphorylation of this reporter. As shown in Fig 4F and G, deletion of LAT1 decreased phosphorylation of Mdh1-GFP, whereas the K75R point mutation drastically increased phosphorylation of the protein by fourfold to 10-fold under these conditions, relative to the phosphorylation level of the reporter in WT control cells. Again, this result supports the conformation-dependent regulation of Pkp1/2 by the PDC, which is decoupled from PDH catalytic activity.
Deletion of PDA1 affects the distribution of Mdh1 in the mitochondrial network, relative to an artificial mtRFP reporter We previously showed that deletion of Aup1, which functions upstream of Pkp2 in the cascade regulating mitophagic selectivity, leads to increased segregation of Mdh1-GFP relative to mtRFP (which does not make native protein-protein interactions with matrix proteins and is therefore not expected to be segregated in the network), within the mitochondrial matrix . This was interpreted as indicating that native proteinprotein interactions between matrix proteins, which are not accessible to the mtRFP reporter, are important in segregating endogenous mitochondrial matrix GFP chimeras into mitophagybound or mitophagy-blocked mitochondrial populations. To test whether deletion of PDA1 similarly affects mitochondrial matrix heterogeneity, we compared the signal overlap between mtRFP and Mdh1-GFP in WT and pda1Δ cells over the first 2 d of incubation in SL medium (Fig 5A and B). WT cells show a slight, statistically insignificant decrease in the signal overlap between day 1 and day 2 of incubation. The pda1Δ mutant, however, showed a statistically significant,~40% reduction in the signal overlap between Mdh1-GFP and mtRFP on day 2 relative to day 1 (P < 0.0001) (Fig 5B), similar to what we previously observed for aup1Δ cells . Importantly, the co-overexpression of Pkp1 and Pkp2 in the pda1Δ cells suppressed the decrease in the signal overlap between days 1 and 2 of incubation. In addition, we observed a mild mitochondrial morphology phenotype in the pda1Δ strain, which appeared on day 2, in which both the red and the green signals appeared to be more clumped into a smaller number of larger foci relative to both WT and kinase-overexpressing strains.
The Lat1 K75R mutation affects physical interactions between the PDC, Aup1, and Pkp1/2 If the PDC affects mitophagy through its physical interactions with Aup1, Pkp1, and Pkp2, we expect that the Lat1 K75R mutant, which has a clear effect on mitophagy, would affect the known physical interactions between Aup1, Pkp1, Pkp2, and the PDC. As shown in Fig 6, we were able to recapitulate the interaction between Aup1 and the PDC under our experimental conditions. We observe that the interaction between Aup1 and Pda1 is weak on day 1 of our experimental timeline, but becomes much stronger on day 3. At the same time, we observe a shift in the mobility of Aup1-HA to slightly faster migration in the day 3 samples, relative to the day 1 samples. Strikingly, the Lat1 K75R mutation abrogates any observable coimmunoprecipitation between Aup1 and Pda1, at both time points. This demonstrates that the conformational change induced by the K75R mutation affects the Aup1-PDC interaction, indicating a potential mechanistic explanation for the up-regulation of Mdh1-GFP mitophagic trafficking and phosphorylation in cells harboring this mutation.

The Lat1 K75R mutation affects protein phosphorylation in the mitochondrial matrix
Our experiments demonstrate the previously undescribed regulation of mitochondrial protein phosphorylation by the PDC, via Pkp1 and Pkp2. Because the Lat1 K75R mutation appears to increase phosphorylation of the Mdh1-GFP reporter, we wanted to determine whether this is a more global effect on protein phosphorylation in the matrix. To this end, WT versus Lat1 K75Rexpressing cells were differentially labeled with heavy and light isotope-substituted arginine and lysine. The cultures were collected on day 3 of incubation in synthetic lactate (SL) medium, the harvested cells were mixed in equal amounts, and mitochondria were enriched by differential centrifugation (Fig 7A and  B). As shown in Fig 7C and in Table S1, we were able to identify at least 21 phosphorylation sites from 19 different mitochondrial matrix proteins, whose phosphorylation state was significantly and reproducibly affected by deletion of PDA1. These results demonstrate that the PDC-mediated regulation of protein phosphorylation in the mitochondrial matrix is not limited to Mdh1. However, the distribution of the results also clearly indicates that the mutation causes both hypophosphorylation and hyperphosphorylation, depending on the phosphorylation site and the specific protein. We therefore suggest that the effect of the PDC on Pkp1-and Pkp2-dependent phosphorylation is not strictly on/off, but rather an effect on kinase selectivity. Interestingly, the proteins that showed significant hyperphosphorylation in the Lat1 K75R mutant included the mitophagy receptor Atg32. In addition, we also identified significant hits in previously undocumented phosphorylation sites in PDC subunits.

Discussion
In this study, we identified novel regulatory interactions between the PDC and its attendant kinases. Although previous studies, from the 1960s to the present, have focused on the fact that PDKs regulate pyruvate dehydrogenase activity (Linn et al, 1969;Gey et al, 2008;Guo et al, 2017a;Cardoso et al, 2020), our data now demonstrate that the PDKs have additional substrates and that their activity toward these non-PDC substrates can be reciprocally regulated by the PDC itself. This regulation may be mediated via allosteric interactions, as it was previously shown that Pkp1 and Pkp2 function as a heterodimer (Gey et al, 2008;Breitkreutz et al, 2010;Walter et al, 2022) and both Aup1 and Pkp1 stably interact with the PDC (Krause-Buchholz et al, 2006;Guo et al, 2017a). Hundreds of mitochondrial matrix proteins have been documented to undergo phosphorylation (Reinders et al, 2007;Kruse & Højlund, 2017;Renvoisé et al, 2017). However, the nature of the signaling cascades involved and their regulation has remained obscure. Our discovery that Pkp1 and Pkp2 can function to phosphorylate mitochondrial matrix proteins other than Pda1 opens up the possibility that at least some of the documented phosphorylation events in the mitochondrial matrix are due to this novel regulatory axis. In support of this, we found by MS analysis that at least 19 additional mitochondrial matrix proteins show differential phosphorylation in response to the lat1 K75R mutation, under our working conditions (Fig  7). Although this list is not comprehensive (many peptides were not identified in all three biological replicates, including the Mdh1 phosphopeptides; see Table S1), it provides an indication for a more general effect of the PDC on mitochondrial protein phosphorylation. We previously showed a functional link between Aup1 and the RTG retrograde signaling pathway (Journo et al, 2009), which transduces stress signals from the mitochondrial matrix to the nucleus (Liu & Butow, 2006), and this may account for the effects on non-matrix proteins such as Atg32. We hypothesize that some of this regulation of mitochondrial protein phosphorylation by the PDC can be ascribed to an allosteric effect of the PDC on Aup1/Ptc6 and Pkp1/2. As Aup1 has been shown to interact with the PDC (Guo et al, 2017a), its phosphatase activity toward other substrates could be influenced by this stable physical interaction. In agreement with this, we can observe the PDC-Aup1 interaction under our conditions, and we further demonstrate that it is strongly affected by the lat1 K75R mutation (Fig 6). Interestingly, the effects of the lat1 K75R mutant on matrix protein phosphorylation are diverse, with some sites being hypophosphorylated and others hyperphosphorylated in cells expressing the mutant, relative to WT cells (Fig 7). This may indicate that the regulation is not a simple on/off switch but rather a change in substrate specificity, suggesting that the association of Pkp1/2 and Aup1 with the PDC regulates substrate specificity, and not simply the activity of the kinases. In support of this, full suppression of the pda1Δ phenotype requires the overexpression of both Pkp1 and Pkp2 (Fig 3C and D), suggesting that multiple phosphorylation (and potentially also dephosphorylation) events are required, in effect altering the physical properties of matrix proteins and their mutual interactions. In addition, we find that the lat1 K75R mutation causes significantly increased phosphorylation of Atg32 (Fig 7 and  Table S1). In agreement with our observation that the lat1 K75R mutant displays increased phosphorylation and mitophagic trafficking of Mdh1-GFP (Figs 2G and 4F), phosphorylation of Atg32 was shown by others to regulate its mitophagy receptor function (Aoki et al, 2011). A missing link in our understanding of the phosphorylationmediated regulation of mitophagic trafficking has been the identity of the opposing phosphatase, which counteracts the function of Pkp1/2 on reporter proteins such as Mdh1-GFP. In the regulation of PDC catalytic activity, it is Aup1/Ptc6, which was shown to dephosphorylate S313 on Pda1 (Guo et al, 2017a). However, the genetic interactions that we uncovered between Aup1 and Pkp2 make it impossible for Aup1 to be playing a similar role in the regulation of mitophagic trafficking. The data shown in Fig 4D and E allows us to suggest Ptc5 as a candidate for a phosphatase, which opposes the phosphorylation of matrix proteins by Pkp1/2. Although further work will be required to determine whether this effect is direct, this result further differentiates the PDC regulatory interactions impinging on pyruvate dehydrogenase activity, from the signaling cascade, which regulates the mitophagic trafficking of individual protein species.
A central result of this study is the discovery of a role of the PDC in regulating matrix protein phosphorylation, independent of its catalytic activity. This is underscored by the fact that the K75R mutation in the E2 subunit Lat1, a mutant that cannot be conjugated to lipoic acid-and is therefore a loss-of-function mutant (Lawson et al, 1991)-actually shows significantly higher levels of phosphorylation and mitophagic trafficking of Mdh1-GFP (see Figs 2G and 4F). Structurally, the lat1 K75R mutation is of great interest, because it forces the PDC into a configuration where most or all E1 subunits carry acetylated TPP, because of a lack of an efficient thiol acceptor. Although this may be an unusual combination under normal physiological conditions, it would be expected under expressing Mdh1-GFP, as well as the indicated plasmids, were grown in SL medium for 5 d, and protein extracts were generated on day 5. The extracts were analyzed by immunoblotting with α-GFP antibodies. (E, F) Quantification of the data in (E). The results of three independent biological repeats were quantified by calculating the % free GFP relative to the total signal and averaging. (G) Expression of Mdh1 T199D -GFP, but not Mdh1 T199A -GFP, bypasses the effect of the pda1Δ mutation. PKY2357 cells expressing Mdh1-GFP, Mdh1 T199D -GFP, and Mdh1 T199A -GFP were grown as described above. Protein extracts were generated on days 1 and 5 and analyzed as above. (G, H) Quantification of the data in (G). All experiments represent an average of at least three independent biological repeats, and statistical analysis was carried out as described in the Materials and Methods section. NS, not significant; asterisks denote significant (P < 0.05) differences. conditions where NAD + (or FAD) is limiting, because a lack of NAD + would prevent regeneration of oxidized lipoic acid. One possible interpretation is that the PDC can function to sense the status of the mitochondrial NAD + pool to regulate matrix protein phosphorylation.
Our data suggest the possibility that the PDC potentially functions as a signaling hub, which regulates protein phosphorylation in the mitochondrial matrix in response to metabolic cues (see the model in Fig 8). One hypothesis that now needs to be tested is that allosteric transitions within the PDC, induced by ligand or substrate (A) Co-overexpression of Pkp1 and Pkp2 suppresses the Mdh1-GFP phosphorylation defect in pda1Δ cells. WT (PKY1089) and pda1Δ (PKY1093) cells expressing Mdh1-GFP, as well as the indicated plasmids, were grown in SL medium for the indicated amounts of time, and protein extracts were generated and immunoprecipitated with α-GFP antibody. Phosphorylation of Mdh1-GFP was detected using α-phosphoamino acid antibody, and total GFP signal in the immunoprecipitate was observed by immunoblotting with the α-GFP antibody. ϕ denotes an empty vector. (A, B) Quantification of the data in (A). The results of three independent biological repeats were quantified by normalizing the α-phosphoamino acid signal to the GFP signal, and averaged. (C) Pkp1 and Pkp2 have a physiological function independent of pda1 phosphorylation. Cells (HAY75 and PKY832) were grown to the late log phase in SD medium and washed, and equal cell numbers were resuspended in SL medium. Cell viability was measured by plating diluted aliquots on rich YPD medium after 5 d of incubation in SL medium. Clearly, the co-overexpression of Pkp1 and Pkp2 completely suppresses the defect in pda1Δ cells (N = 3; error bars denote the SD), * denotes P < 0.05 (t test). NS, not significant. (D) Deletion of PTC5 partially suppresses the pda1Δ mitophagy defect. Mdh1-GFP-expressing WT (PKY974), pda1Δ, and pda1Δ cells harboring co-deletions of PTC7 (PKY2234), PTC5 (PKY2239), or both (PKY2286) were incubated in SL medium for 5 d, and protein extracts were generated. The extracts were analyzed by immunoblotting with α-GFP antibodies. (D, E) Quantification of three independent biological repeats of the experiment shown in (D). (F) Catalytically inactive Lat1 K75R mutant causes massive hyperphosphorylation of Mdh1-GFP. Mutant lat1Δ cells (PKY2298) expressing the indicated plasmids, as well as Mdh1-GFP, were grown in SL medium for the indicated amounts of time, and protein extracts were generated and immunoprecipitated with α-GFP antibody. Phosphorylation of Mdh1-GFP was detected and quantified as in (A, B). (F, G) Quantification of the data in (F). All experiments represent an average of at least three independent biological repeats, and statistical analysis was carried out as described in the Materials and Methods section. NS, not significant; asterisks denote significant (P < 0.05) differences. binding, will affect the structure of the complex, leading to changes in the activity of its associated kinases and phosphatase(s). The entire system, including the redundancy of the pyruvate dehydrogenase kinases (there are four PDKs in mammals) and the attendant PDC phosphatase, seems to be highly conserved. Strikingly, the region around the yeast Mdh1 phosphorylation site at Thr199 (IGGHSGITIIPPLISQ), which we previously showed to be crucial for mitophagic trafficking, is also highly conserved in human MDH2 (IGGHAGKTIIPLISQ) and the homologous Thr204 in the human protein was also reported to be phosphorylated (Kettenbach et al, 2011). Therefore, it is likely that these reciprocal regulatory interactions are also conserved. Indeed, elegant biochemical studies from the Roche laboratory demonstrated that the activation of human PDK2 by NADH and acetyl-CoA was due to bound E2 Figure 5. Deletion of PDA1 affects the intra-mitochondrial distribution of Mdh1-GFP. (A) Deletion of PDA1 affects distribution of Mdh1-GFP within the mitochondrial matrix. Cells (PKY974 and PKY2023) co-expressing Mdh1-GFP from the endogenous MDH1 promoter and mtRFP (which does not make native protein-protein interactions with matrix proteins and is therefore not expected to be segregated within the network) were imaged on days 1 and 2 of incubation (to avoid interference from the vacuolar signal from day 3 and up), and the overlap between the red and green channels was quantified as described in the "Materials and Methods section." White arrows indicate individual examples of differential distribution of fluorescence between the two channels. (B) Graph showing quantification of the overlap between the two channels in WT and in pda1Δ cells, and indicating statistical significance (NS, not significant). Results are representative of three biological replicates. Scale bar = 5 μm.
The PDC regulates phosphorylation of mitochondrial proteins Kolitsida et al.
https://doi.org/10.26508/lsa.202302149 vol 6 | no 9 | e202302149 9 of 15 becoming acetylated in the partial reverse reaction, demonstrating the existence of this exact type of allosteric coupling in human PDKs (Baker et al, 2000), although in that case, their conclusions limited the implication to the allosteric regulation of E1α phosphorylation.
Our results now suggest a different interpretation for these previous findings, wherein allosteric shifts in the E2 may regulate the phosphorylation of additional substrates. Similarly, it was recently reported that the overexpression of PDK4 enhances mitophagy in mammalian cells (Park et al, 2015), although again the assumption was that this reflects the regulation of pyruvate dehydrogenase activity. Another recent study found that branched-chain amino acids allosterically modulate PDC catalytic activity (Li et al, 2017), supporting the notion that conformational changes in the PDC occur in response to metabolic cues. The results we have presented here suggest an additional dimension to the picture, by indicating that the PDC functions upstream of the PDKs to regulate mitochondrial function in response to such conformational changes. The concept of large signaling complexes that integrate metabolic cues to regulate cellular activity has been the focus of intense study in recent years. TORC1 and TORC2 are examples of such mega- Figure 6. Lat1 K75R mutation affects the physical interaction between Aup1 and the PDC. Cells deleted for LAT1 (PKY2498) and expressing Aup1-HA and Pda1-FLAG, as well as the indicated LAT1 variant (empty vector, WT, or K75R), were incubated in SL medium for 1 or 3 d as indicated, and samples were analyzed by immunoprecipitation with α-HA antibodies and immunoblotted for HA or FLAG epitopes as indicated. complexes, which integrate nitrogen, amino acid, and carbohydrate metabolism with hormone signaling, to generate readouts that regulate cellular responses to changes in the environment (Eltschinger & Loewith, 2016;Nicastro et al, 2017;Szwed et al, 2021). We hypothesize that the PDC could serve a similar function within mitochondria, by regulating matrix protein phosphorylation in response to changes in metabolite levels. Further work will now be required to test specific aspects of this hypothesis.

Materials and Methods
Yeast strains, plasmids, and growth conditions Yeast strains used in this study are listed in Table S2. Deletion mutants and epitope-tagged strains were generated by the cassette integration method (Longtine et al, 1998). Strain HAY75 (MATα leu2-3,112 ura3-52 his3-Δ200 trp1-Δ901 lys2-801 suc2-Δ9) was used as the WT genetic background in this study.
All knockout strains were verified by PCR. Oligonucleotides used in this study are detailed in Table S3, and plasmids are detailed in Table S4.
For nitrogen starvation experiments, cells were grown to the mid-log phase (OD 600 of 0.4-0.6) in synthetic dextrose (SD) medium, washed with distilled water, and resuspended in nitrogen starvation medium (0.17% yeast nitrogen base lacking amino acids and ammonium sulfate [Difco], and 2% glucose) for the times indicated in individual experiments. Overexpression studies with CUP1 promoter-based vectors were carried out by supplementing the medium with 5 μM CuSO 4 , for both control (empty vector) and overexpressing cells.

Chemicals and antisera
Chemicals were purchased from Sigma-Aldrich unless otherwise stated. Custom oligonucleotides were from Hylabs. Anti-GFP antiserum was from Santa Cruz. Horseradish peroxidase-conjugated goat anti-rabbit antibodies were from MP Biomedicals. Antiphosphoserine/threonine antibodies were from ECM Biosciences (Cat. No. PM3801).

Preparation of whole-cell extracts for Western blot analysis
Cells (10 OD 600 units) were treated with 10% cold trichloroacetic acid and washed three times with cold acetone. The dry cell pellet was then resuspended in 100 μl cracking buffer (50 mM Tris, pH 6.8, 6 M urea, 1 mM EDTA, and 1% SDS) and vortexed in a Heidolph Multi Reax microtube mixer at maximum speed with an equal volume of acid-washed glass beads (425-600 }m diameter), for a total of 30 min. Lysates were clarified by centrifugation at 17,000g for 5 min, and total protein was quantified in the supernatant using the BCA protein assay (Thermo Fisher Scientific). SDS loading buffer (final concentrations of 100 mM Tris, pH 6.8, 20% glycerol, 2% SDS, and 500 mM β-mercaptoethanol) was added to the lysate, and the samples were warmed to 60°C for 5 min before loading on gels. ImageJ software was used for band quantification.
Free GFP release assays for analyzing mitophagic trafficking efficiency Quantitative analysis of mitophagic trafficking was analyzed using GFP chimeras of endogenous mitochondrial matrix proteins or an artificial mitochondrially targeted GFP chimera of mouse cytoplasmic DHFR. This assay relies on the resistance of GFP to proteolytic degradation in the yeast vacuole relative to that of other proteins. Because GFP molecules derived from different chimeras can be expected to have the same lifetime, this allows a semiquantitative analysis of the efficiency of mitophagic targeting as measured by the percentage of the total signal that is converted to free GFP (Klionsky et al, 2016;. Immunoblotting SDS-10% polyacrylamide gels were transferred to a nitrocellulose membrane using either a wet or a semidry blotting transfer system. Membranes were blocked for 1 h with TPBS (137 mM NaCl, 2.7 mM KCL, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , 1% Tween, and 5% milk powder [BD Difco Skim Milk]), followed by incubation with rabbit anti-GFP (1: 5,000) for 1.5 h, then washed 4 × 2 min with TPBS, and incubated for 1.5 h with HRP-conjugated secondary antibody (1:10,000). The membranes were then washed 4x with TPBS, incubated with the SuperSignal Chemiluminescence substrate (Thermo Fisher Scientific), and exposed to a Molecular Imager ChemiDoc XRS imaging system. Where necessary, 2% normal goat serum was added to blocking solutions to reduce background. We propose that the known stable physical interactions between Aup1 and Pda1 (Guo et al, 2017a) and between Pkp1 and Pda1 (Krause-Buchholz et al, 2006) can function as an allosteric interface to regulate Aup1, Pkp1, and Pkp2 activity toward other proteins within the mitochondrial matrix. We previously showed that Pkp2 functions downstream of Aup1, and in this study, we showed that the cooverexpression of Pkp1 and Pkp2 completely overcomes the mitophagy defect in pda1Δ cells. Because we also demonstrate that the mitophagy-related function of Aup1 is independent of Pda1 phosphorylation, we propose that Pda1-bound Aup1 functions downstream of Pda1 to transduce signals to the kinase(s) in the pathway. It remains to be seen whether physiological PDC ligands can allosterically regulate phosphorylation in the matrix.
Calculation of statistical significance of differences in % free GFP signals from immunoblots Data (% free GFP) were log-transformed before analysis to stabilize variances. A repeated-measures ANOVA was performed to compare mutants and proteins simultaneously. Thereafter, mutants were compared for each protein and proteins were compared for each mutant by the Tukey HSD test (P < 0.05). ANOVA was carried out using JMP 12 software.

SILAC labeling
To quantify relative phosphorylation on mitochondrial proteins in WT versus pda1Δ backgrounds, cell cultures of each strain were grown separately in SL medium supplemented respectively with 0.003% (wt/vol) 13 C 6 15 N 2 L-lysine (Lys8; Cat. No. 211604102; Silantes) and 0.001% (wt/vol) 13 C 6 15 N 4 L-arginine (Arg10; Cat. No. 201603902; Silantes) or with identical concentrations of unlabeled lysine and arginine, in addition to the standard auxotrophy supplementation. At the indicated time point, 50 OD 600 units of cells from each labeling regime were combined and washed with cold lysis buffer, and cells were frozen in liquid nitrogen. To lyse the cells, the frozen suspensions were processed in a SPEX Freezer/Mill according to the manufacturer's instructions. The frozen ground powder was thawed and clarified by centrifuging for 10 min at 500g. The clarified lysates were then further centrifuged for 10 min at 13,000g, the supernatant was discarded, and the pellet was precipitated with 10% ice-cold trichloroacetic acid and washed 3x with cold acetone.

MS analysis
Mass spectrometric measurements were performed on a Q Exactive HFX mass spectrometer coupled to an EasyLC 1200 (Thermo Fisher Scientific). Before analysis, phosphopeptides were enriched on Automated Liquid Handling Platform (AssayMap Bravo; Agilent) using Fe(III)-NTA cartridges (5 μl) essentially as described previously (Post et al, 2017). The MS raw data files were analyzed by MaxQuant software (Cox & Mann, 2008), version 1.4.1.2, using a UniProt Saccharomyces cerevisiae database from March 2016 containing common contaminants such as keratins and enzymes used for in-gel digestion.

Statistical analysis
Phosphosite SILAC ratios were normalized to respective protein changes to discriminate regulated sites from regulated proteins. Measurements of three biological replicates (Pearson's correlation of 0.64-0.71) comparing WT with pda1Δ cells were averaged and log 2 -transformed. To determine sites that changed significantly between genotypes, we calculated an outlier significance score on the merged log 2 -transformed phosphosite ratios according to Cox & Mann (2008) (significance A, P < 0.05, BHcorrected).
For statistical analysis of Western blotting quantifications in Figs 3B and 4B and G, % normalized phospho-AA signal intensity values relative to GFP signal values were calculated from raw files. M-ANOVA was carried out for an overall comparison of all the genotypes measured on 3 d (day 1, day 3, and day 5; N = 3). Log transformation of the data was applied before analysis to stabilize variances. A pairwise comparison of genotypes was carried out by contrast tests.

Site-directed mutagenesis
Site-directed mutagenesis protocol was carried out according to the Stratagene QuikChange system. A Pfu DNA polymerase (Fermentas) was used with a thermocycling protocol followed by removal of the parental strands with DpnI digestion. Thermocycling conditions were as follows: initial denaturation at 95°C for 2 min, then (steps 2-4) 30 s at 95°C, 60 s at 55°C, and 12 min at 68°C. Steps 2-4 were repeated for 18 cycles. PCR products were treated with DpnI (NEB) for 1 h at 37°C and precipitated with 70% ethanol. DNA pellets were then dried and resuspended with 20 μl TE buffer, pH 8. All mutagenesis products were transformed into E. coli DH5α, and mutations were verified by sequencing. The oligonucleotide primers used to create site-directed MDH1-GFP variants and combined variants are listed in Table S3.

Fluorescence microscopy
Culture samples (3 μl) were placed on standard microscope slides and viewed using a Nikon Eclipse E600 fluorescence microscope equipped with the appropriate filters and a Photometrics CoolSnap HQ CCD. To achieve statistically significant numbers of cells per viewing field in the pictures, 1 ml cell culture was collected and centrifuged for 1 min at 3,500g before sampling. For quantitative analysis of co-localization, ImageJ software with the co-localization plugin, JACoP (Bolte & Cordelières, 2006), was used. Representative fields were analyzed in terms of channel intensity correlation coefficient.
For confocal microscopy, cells were placed on standard microscope slides, and micrographs were obtained by confocal laser scanning microscopy using a Leica SP8, using a 63x water immersion lens. GFP excitation was carried out using the 488-nm line, and emission was collected between 500 and 540 nm. For RFP excitation, we used the 552-nm line and fluorescence emission was collected between 590 and 640 nm. Deconvolution of Z-stacks was carried out using ImageJ software.
For statistical analysis of channel overlap data, ANOVA was carried out using JMP 16.0 software. Correlations were compared over mutations and days by two-factor ANOVA. As the mutation X day interaction effect was statistically significant (P < 0.0001), the 2 d were compared for each mutation by contrast t tests. Preplanned comparisons with WT were also performed by contrast t tests.

Determination of colony-forming efficiency
Cells (HAY75 and PKY832), harboring empty vectors or plasmids overexpressing PKP1 and PKP2, were grown to the late log phase in SD medium and washed, and equal cell numbers were resuspended and incubated in SL medium for 5 d. After 5 d, the cells were recounted, diluted to an expected confluency of 175 colonies/plate, and plated on YPD plates (see Fig S5 for an example).